Cascade Reactions Enabled by Synthetic Biology

Nicholas J. Turner School of Chemistry & Manchester Institute of Biotechnology,

University of Manchester, UK

ELRIG Research & Innovation 2016 Nottingham, UK 23rd March 2016

Manchester Institute of Biotechnology Discovery through innovation

Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals

Nigel Scrutton (PI/Director) Eriko Takano (Director) Nick Turner (Director)

Manchester Institute of Biotechnology Discovery through innovation

CoE for Biocatalysis, Biotransformations and Biocatalytic Manufacture (est. 2005).

C M B C Manchester Centre for Biophysics and Catalysis (est. 2009).

Manchester Centre for Integrative Systems Biology (est. 2006).

CENTRES

BBSRC IB NETWORKS (est. 2014) Network in Biocatalyst Discovery, Development and Scale-up. Glycoscience Tools for Biotech. and Bioenergy Network.

Natural Products Discovery and Bioengineering Network.

BBSRC/EPSRC SYNBIOCHEM Centre for Synthetic Biology of Fine and Speciality Chemicals (est. 2014).

EPSRC National Biocatalysis and Biotransformations Hub (coordinated by MIB/Harwell) (est. 2015).

NEW CENTRES

SYNBIOCHEM Approach

Biocatalyst/Pathway Selection

design modify

homologues

Pathway design Vector promoter

RBS Gene cluster order

Chassis engineering design modify

homologues Eg. Carbon source, minimal genome

Pathway assembly

synthesis automated assembly

TEST

Predictive modelling metabolism kinetic/flux

catalysis machine learning

CAD tools

Regulatory devices light activation

riboswitches circuit design

Genome-editing tools

Speedy Genes

Testing targeted analysis

untargeted metabolomics Microfluidics

Screening assays

High Throughput Tools

(Robotics, microscale growth/expression,

analysis/assays)

State of the Art analytics facility

Scale-up Fermentation optimisation

Compilation and assembly tools

Jr ICE Gene Genie

Fully integrated technology platforms

3 Approaches to (un)natural product synthesis

Biosynthesis Organic

Synthesis Biocatalysis

• Natural products • Biosynthetic pathways • Enzyme mechanism • Specialised enzymes

• New reagents • New catalysts • Retrosynthesis • Synthetic strategy

• Engineered biocatalysts • Broad specificity • Systems biocatalysis • Synthetic biology

Nature Chem. Biol., 2013, 9, 285-288.

Biocatalytic retrosynthesis

HN

NH

N

H

H HN

HN

O

O

OO

ON

NO

Telaprevir (Incivek)

launched

May

2011

for

Hepatitis

C

NH

N

biocatalyst

99% e.e.

• Design new & general synthetic routes to target classes (e.g. amino acids, alkaloids, terpenes etc.) based upon bio- and chemo-catalysis?

• Develop guidelines for route design for synthetic chemists

(biocatalytic retrosynthesis).

• Where are the gaps in biocatalysis – which reactions are currently not available in the biocatalysis toolbox?

• How many different biocatalyst classes do we need to be able

to do real organic synthesis? 50 or 500 or 5000 … • Need biocatalysts with broad substrate scope that are active

and stable under the conditions of a chemical process (fit biocatalyst to process rather than vice-versa).

Challenges for biocatalysis

Protein Design Biocatalysts Synthesis Protein Evolution

Design – Evolution - Diversity

NH

NH

Design features: • Selectivity • Specificity • Stability

HN

Natural Diversity

Alkaloids

NH

N

(R)-harmicine(anti-leishmania)

H

N

MeO

MeOH

Crispine A(anti-tumour)

NH

NH

Me

(R)-Eleagnine(chocolate, cocoa)

H

NH

(R)-coniine(hemlock)

N

NMe

(S)-nicotine

(S)-scoulerine

N

MeO

HOOH

OMe

N

MeO

HOOH

OMe

(S)-reticuline

NH

MeO

MeO

(R)-salsolidine

Synthetic APIs

Solifenacin Levocetirizine

Telaprevir

N

ClN

OOH

O

H NO

ON

N

NNH

O HN

ON

O

H

H

O NHO

O NHN CO2H

OHN

SO ONH

HN

H2N NH

Argatroban

(Asymmetric) biocatalytic amine toolbox

R1

O

R2 R1

NH2

R2

transaminase

PLP

R1

O

R2 R1

NH2

R2

aminedehydrogenase

NADH

R1

NH2

R2 R1

NH

R2

amine oxidase

FAD

ArCO2H

ammonia lyaseAr

CO2H

NH2

NH

R NH

R

imine reductase

NADPH

NH+

R

NH

R

Pictet-Spenglerase

X X

NH

NOpine DH

NADPH

OR+

R

Biocatalytic Cascade Reactions

Regio- and stereoselective ω-transaminase/MAO-N cascades

ORL

ORS N RLRS

NH

RLRS

NH

RLRS

(R)(S)

(S)(S)

+

Accumulates

NH3.BH3non-selective reduction

MAO-N oxidation

(S)

(S)-selectiveomega-TA

Regio- & stereoselectivereductive-amination

Regio- & stereoselectiveoxidation

One-potnon-symmetric diketone

E. O’Reilly et al., Angew. Chem. Int. Ed., 2014, 53, 2447-2450.

O

O

N(S )-

C. viola

ceum

transam

inase

MAO-N D9BH3NH3

NH

ee: >99% (S) de: >99%(2R,5S)

ω-TA - MAO-N tandem reaction

E. O’Reilly et al., Angew. Chem. Int. Ed., 2014, 53, 2447.

N

(R)-Arthrobacter

transaminase MAO-N D9BH3NH3 N

Hee: >99% (R) de: 90%(2R,5R)

Cascade reactions with TAs/IREDs

NH

R1R2IRED

N R1R2-H2O

R1

O

R2

NH2ω-TA

R1

O

R2

O

2,6-disubstituted

piperidine

1,5-diketone

O

O

Nω-TA

(R)- or (S)-IRED

NH

ee: >99% (S)

Oω-TA

(R)- or (S)-IRED

ee: >99% (S)

O

N NH

X X X

ω-TA - IRED tandem reactions

Shahed Hussain, Elaine O’Reilly.

Cascade reactions with IREDs

NH

R1R2IRED

N R1R2-H2O

R1

O

R2

NH2ω-TA

R1

O

R2

O

2,6-disubstituted

piperidine

1,5-diketone

N R1

R2

IRED -H2Oω-TA

1,5-ketoaldehyde

NH

R1

R2

R1

ONH2R2

O

R1

OR2

HCAR

HO

O

R1

OR2

1,5-ketoacid

CAR - ω-TA - IRED tandem reactions

H

O

O

Nω-TA

(R)- or (S)-IRED

NH

ee: >99% (S)

n

n n

HO

O

O

n

carboxylic acidreductase, ATP, NADH

Elaine O’Reilly, Shahed Hussain, Scott France, Andy Hill.

O’Reilly et al., Angew. Chem. Int. Ed. 2014, 53, 10714 - 10717 (VIP).

ONH2

ω-TAN

H

NH2NH2

NH

polyisoindole(coloured)

irreversible amine donor

(S)-scoulerine

N 1. MAO-N

D11:

NH3:BH3

2. Berberine

Bridge

Enzyme

yield = 97%

e.e. = 99%

MeO

HOOH

OMe

N

MeO

HOOH

OMe

(R/S)-reticuline

N

R1

R1

R1

R1

e.e.'s > 98%

MAO-N/Berberine bridge enzyme

J. Schrittwieser, D. Ghisleri, W. Kroutil, N.J. Turner et al., Angew. Chem. Int. Ed., 2014, 53, 3731-3734.

Biocatalytic asymmetric hydrogen borrowing

F.G. Mutti. T. Knaus, N.S. Scrutton, M. Breuer and N.J. Turner, Science, 2015, 349, 1525-1529.

R R'

O

R R'

NH2

R R'

OH

*

NADH

NAD+

ADH AmDH

NH3 / NH4+

H2Ocatalytic

OH (S)-ADH

(R)-AmDH

NH3 (2.5M)

NADH (1 mol%)conv. 95%ee: >99% (R)

FNH2

F

OH (R)-ADH

(R)-AmDH

NH3 (2.5M)

NADH (1 mol%)conv. 95%ee: >99% (R)

FNH2

F

Biocatalytic asymmetric hydrogen borrowing

OH

R'X

X = H, F, Me, MeO; R' =

Me, Et

yields = 30-95%e.e.'s up to 99%

X

X = CH2 or O

yields = 84-96%e.e.'s up to 99%

X

X = H, F, Me

yields = 7-96%e.e.'s up to 99%

Alkyl

OH

Me

Alkyl = n-C6H13, n-C5H11. n-

C4H9, n-C3H7, iso-C4H9

yields = 66-96%e.e.'s >99%

R = n-C7H15, n-C6H13, n-C5H11n-C4H9, iso-C4H9, n-C3H7, PhCH2

yields = 8-99%

Me

OH

Me

OH

OHR

F.G. Mutti. T. Knaus, N.S. Scrutton, M. Breuer and N.J. Turner, Science, 2015, 349, 1525-1529.

Whole-cell biocatalysts for stereoselective C-H amination reactions

P. Both et al, Angew. Chem. Int. Ed., 2015, 54, in press.

Acknowledgements

amine biocatalysis:

Rachel Heath, Marta Pontini, Friedemann Leipold, Shahed Hussain, Godwin Aleku, James Galman, Anthony Green,

Elaine O’Reilly, Beatrice Bechi, Scott France, Iustina Slabu, Andy Hill, Fabio Parmeggiani, Syed Ahmed, Nick Weise,

Wojciech Zawodny, Agata Brzezniak, Francesco Mutti, Diego Ghislieri, Juan Mangas

and everyone else in the group …

Sam Staniland, Sasha Derrington, Chantel Jensen-

Loughrey, Will Birmingham, Ian Rowles, Mark Corbett, Jane Kwok, Frank Xu, Mark Dunstan, Daniela Quaglia

Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Whole-cell biocatalysts for stereoselective C-H amination reactionsSlide Number 23